The present invention relates generally to cryogenic preservation, and more particularly to cryogenic preservation of biologically active materials using vitrification techniques.
Cryogenic preservation (cryopreservation) can be defined as lowering the temperature of living structures and biochemical molecules to the point of freezing and beyond, for the purposes of storage and future recovery of the material in its pre-frozen, viable condition. Experiments with canine sperm in the 1700""s first demonstrated that single cells could be frozen and later thawed, and that a small percentage of cells returned to normal physiological function. Later, in the 1900""s, it was found that cell recovery rates could be improved if the cells where chemically prepared to withstand the freezing process using compounds collectively referred to as cryoprotectants. However, even with the use of cryoprotectants, recovery rates from cryopreservation are routinely 50 percent or less.
To date attempts to improve cryopreservation recovery rates have generally focused on new cryoprotectants to treat biological material prior to freezing and extremely slow or fast freezing techniques. Both techniques are generally directed towards reducing cellular damage caused when the water within cells expands due to the formation of ice crystals during the freezing process. In theory, extremely slow or fast freezing will reduce or eliminate the formation of ice crystals within a cell. Mechanisms for extremely slow rates of freezing have included controlled descent through nitrogen vapors into liquid nitrogen, or moving samples through super-cooled alcohol compounds followed by plunging into liquid nitrogen. Freezing in this manner does not allow further growth of ice crystal size during the freezing process, but still allows ice crystal formation.
Another technique, often referred to as vitrification, plunges samples directly into liquid nitrogen in an attempt to freeze the water within the cell so rapidly that ice crystal formation is inhibited. Vitrification rapidly takes cells from room temperature to xe2x88x92196xc2x0 C., the temperature of liquid nitrogen. Such an extreme drop in temperatures in such a short time often causes stress fractures within the cell membrane. Cryoprotectants are used in conjunction with vitrification and various other freezing techniques.
Therefore, what is needed is an improved way to cryogenically preserve viable single cells, tissues, organs, nucleic acids, or other biologically active molecules, that avoids at least some of the problems inherent in currently available methods. Accordingly, at least one embodiment of the present invention provides a method of cryopreservation comprising immersing biologically active material in cooling fluid and circulating the cooling past the material. The material may or may not undergo chemical preparation prior to immersion, depending on the type of materials being cryopreserved. The cooling fluid is circulated past the material at a substantially constant predetermined velocity and temperature to freeze the material such that the material is vitrified, and the formation of stress fractures in cell membranes is minimized. In at least one embodiment, the cooling fluid is maintained at a temperature of between about xe2x88x9220 degrees centigrade and xe2x88x9230 degrees centigrade, and the velocity of the cooling fluid past the material is about 35 liters per minute per foot of cooling fluid through an area not greater than about 24 inches wide and 48 inches deep. Additionally, at least one embodiment of the present invention freezes the material directly to a temperature higher than about xe2x88x9230 degrees centigrade. Yet another embodiment of the present invention provides a biological material having been subjected to such a cryopreservation process.
An object of at least one embodiment of the present invention is to freeze biological material such that the formation of ice crystals and stress fractures are avoided.
An advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are significantly increased, because biological material is vitrified during freezing.
Another advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are improved, because biological material is vitrified at a high enough temperature to avoid the formation of stress fractures within cell membranes.
A further advantage of at least one embodiment of the present invention is that once frozen, current cryoperservation storage facilities and mechanisms can be used to store the frozen biological material.